Side-chain oxidized oxysterols as metabolic regulators in liver and brain

Oxysterols are oxygenated derivatives of cholesterol characterized by a very short half-life and their ability to pass lipophilic membranes easily, thus they are considered as important intermediates in cholesterol excretion pathways and its conversion to bile acids. The two major oxysterols in the circulation of human and mouse are 24S-hydroxycholesterol (24S-OH) and 27-hydroxycholesterol (27-OH), which are formed by the cytochrome P450 enzymes cholesterol 24-hydroxylase (CYP46A1) and sterol 27-hydroxylase (CYP27A1), respectively. The two oxysterols 27-OH and 24S-OH are both strong inhibitors of cholesterol synthesis and activators of LXR in vitro. However, their role as physio- logical regulators under in vivo conditions is controversial. The overall aim of this thesis was to investigate the regulatory role of side chain oxidized oxysterols as metabolic regulators in vivo. In particular we have studied the role of 24S-and 27-hydroxycholesterols (24S- and 27-OH) as regulators of cholesterol synthesis and activators of LXR. We used mouse models with increased levels of 27-OH (CYP27A1) transgenic mice and Cyp7b1 knock-out mice (Cyp7b1-/-) as well as a mouse model with no detectable levels of 27-OH in their circulation, Cyp27a1 knock-out mice (Cyp 27-/-). The latter mice were treated with cholic acid to compensate for the reduced formation of bile acids. In Paper I, we studied a possible regulatory role of 27-OH and 24S-OH in the brain using human CYP27A1 transgenic mice(CYP27A1tg) and Cyp27a1 knock-out (Cyp 27a1-/-) mice. The levels of 27-OH were increased about 12-fold in the brain of CYP27A1tg mice while the levels of 24S-OH was decreased by about 25%, most probably due to increased metabolism by the CYP27A1 enzyme. The mRNA levels of HMG-CoA reductase and HMG-CoA synthase in the brain were increased. In accordance with increased cholesterol synthesis, most of cholesterol precursors were also increased. The increased cholesterol synthesis is likely due to reduced inhibition by 24S-OH. 27-OH is an activator of LXR and in spite of this, there was no upregulation of the LXR-target genes in the brain of the transgenic mice. In contrast, some of the genes were downregulated. In Cyp27a1-/- mouse brain, cholesterol synthesis was slightly increased with increased levels of cholesterol precursors. The increased synthesis is probably the consequence of the absence of an inhibitory effect of the flux of 27-OH into the brain. The results of this study are consistent with the possibility that both 24S-OH and 27-OH have a suppressive effect on cholesterol synthesis in the brain. Since there was no activation of the LXR-target genes in the brain of the transgenic mice, we concluded that 27-OH is not a general activator of LXR in the brain. In Paper II, this study has examined the role of 27-OH in the liver using the above three mouse models. In the liver of CYP27A1tg mice we found a modest increase of the mRNA levels corresponding to the LXR target genes Cyp7b1, and Abca1. There was no effect on a number of other LXR-regulated genes. There were no significant effects on cholesterol synthesis at the transcriptional level and cholesterol precursors were not affected as well. However, there was a modest decrease in T-MAS levels in the liver of CYP27A1tg mice. In the liver of the Cyp7b1-/-mice, there were also no effects on cholesterol synthesis neither at the transcriptional level nor in the levels of cholesterol precursors, with the exception of increase in desmosterol. In connection to the LXR-target genes in these mice, there were no differences in the expression between the Cyp7b1-/- and the wild type mice. If the high levels of 27-OH are important, the same effects would be expected in the two mouse models. In the liver of the Cyp27a1-/- mice there was a slight activation of some LXR- regulated genes, Abcg5, Abcg8, Fas and Srebp1c. If 27-OH is of importance as a normal activator of the above genes a suppressing effect would be expected. The overall results do not support the contention that 27-OH is an important regulator of cholesterol homeostasis or an activator of LXR-regulated genes under basal conditions in the liver. In conclusion our results suggest that both 24S-OH and 27-OH may be of some regulatory importance for cholesterol synthesis in the brain but not in the liver. Under normal basal conditions 27-OH does not seem to be a general activator of LXR neither in the brain nor in the liver. The different effects on cholesterol synthesis in the two organs may be related to the fact that almost all oxysterols in the brain are in the free form whereas most of them are esterified in the liver

Side-chain oxidized oxysterols as metabolic regulators in vivo

Oxysterols are oxygenated derivatives of cholesterol characterized by a very short half- life and their ability to pass lipophilic membranes easily, and they are considered as important intermediates in the excretion pathways of cholesterol and its conversion to bile acids. Evidence has been presented that the production and flux of oxysterols in the brain may also be of some importance for cognitive functions. The two major oxysterols in the circulation of human and mouse are 24S-hydroxycholesterol (24S- OH) and 27-hydroxycholesterol (27-OH), which are formed by the cytochrome P450 enzymes cholesterol 24-hydroxylase (CYP46A1) and sterol 27-hydroxylase (CYP27A1), respectively. The two oxysterols 27-OH and 24S-OH are both strong inhibitors of cholesterol synthesis and activators of LXR in vitro. However, their role as physiological regulators under in vivo conditions is controversial. The overall aim of this thesis was to investigate the regulatory role of side-chain oxidized oxysterols as metabolic regulators in vivo. In particular, we have studied the role of 24S-OH and 27-OH as regulators of cholesterol synthesis and activators of LXR. We used mouse models with increased levels of 27-OH like human CYP27A1 overexpressor mice (CYP27A1tg) and Cyp7b1 knockout mice (Cyp7b1-/-), as well as a mouse model with no detectable levels of 27-OH in the circulation, Cyp27a1 knockout mice (Cyp 27a1-/-). The latter mice were treated with cholic acid to compensate for the reduced formation of bile acids. Using Cyp27a1-/- mice, we have also studied the possibility that 27-OH mediates the negative effects of dietary cholesterol on memory function in mice. In Paper I, we studied a possible regulatory role of 27-OH and 24S-OH in the brain, using CYP27A1tg mice and Cyp27a1-/- mice. The levels of 27-OH were increased by approximately 12-fold in the brain of CYP27A1tg mice while levels of 24S-OH were decreased by about 25%, most probably due to increased metabolism by the CYP27A1 enzyme. Evidence was presented that cholesterol synthesis was increased in the brain of the two mouse models. There was no upregulation of the LXR-target genes in the brain of either of the two models. The increased synthesis in the brain of the Cyp27a1-/- mice is probably the consequence of the absence of an inhibitory effect of the flux of 27-OH into the brain. The increased cholesterol synthesis in the brain of the CYP27A1tg mice is probably due to the reduced levels of 24S-OH, leading to reduced inhibition of its synthesis. The results of this study are consistent with the possibility that both 24S-OH and 27-OH have a suppressive effect on cholesterol synthesis in the brain. We also conclude that 27-OH is not a general activator of LXR in this tissue. In Paper II, we examined the role of 27-OH in the liver using the three mouse models described above. Only very modest effects on cholesterol synthesis and LXR target genes were observed in the three mouse models. The overall results do not support the contention either that 27-OH is an important regulator of cholesterol homeostasis, or that 27-OH is an activator of LXR-regulated genes under basal conditions in the liver. It has been reported that treatment of mice with dietary cholesterol leads to upregulation of some LXR target genes in the liver. In a study by Chen et al, such upregulation of three different LXR target genes was not seen when treating mice lacking 24S-OH, 25-OH and 27-OH with dietary cholesterol (Chen et al., 2007). It was concluded that a 24-, 25- or 27-hydroxylation step mediated the effect of dietary cholesterol. We show here that similar effects were obtained when treating Cyp27a1-/- mice with dietary cholesterol, suggesting that the effects are mediated by 27-OH. Most of these effects could however not be observed at either the protein level, or at the level of enzyme activity. The results suggest that 27-OH is a mediator of the cholesterol-induced effects on some LXR target genes when the mice are challenged with a high load of dietary cholesterol. The physiological importance of this effect is difficult to evaluate. In Paper III we compared the two mouse models CYP27A1tg and Cyp7b1-/- mice, both of which have high levels of 27-OH in the circulation and the brain. In contrast to CYP27A1tg mice, the levels of 24S-OH in the brain were not decreased in the latter model, and cholesterol synthesis was not affected. This supports the proposal that 24S- OH is of regulatory importance for cholesterol synthesis in the brain. It has been reported that cholesterol synthesis is reduced in the kidney of Cyp7b1-/- mice. Cholesterol synthesis in the kidney of CYP27A1tg mice was not affected, however. We conclude that factors other than high levels of 27-OH are of importance for the reduced cholesterol synthesis in the kidney of the Cyp7b1-/- mice. In Paper IV we tested the possibility that the negative effect of dietary cholesterol on cognition in mice is mediated by 27-OH. The negative effect of dietary cholesterol on spatial memory observed in wild-type mice was not observed in Cyp27a1-/- mice. The latter mice were also treated with cholic acid to compensate for the reduced synthesis of bile acids. Treatment with dietary cholesterol was shown to lead to reduced levels of the “memory protein” Arc (Activity-Regulated Cytoskeleton-associated protein) in the hippocampus of the wildtype mice. This effect was not seen in the hippocampus of Cyp27a1-/- mice. The results are consistent with the possibility that 27-OH is a mediator of the negative effects of dietary cholesterol on cognition. In conclusion, our results suggest that both 24S-OH and 27-OH may be of some regulatory importance for cholesterol synthesis in the brain but not in the liver. Under normal basal conditions 27-OH does not seem to be a general activator of LXR, either in the brain or in the liver. We discuss the possibility that the different effects on cholesterol synthesis in the two organs may be related to the fact that almost all oxysterols in the brain are in the free form, whereas in the liver, most of them are esterified. In addition, we demonstrate that the negative effects of dietary cholesterol on cognition is mediated by 27-hydroxylation

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Bacterial etiology of community-acquired pneumonia in immunocompetent hospitalized patients and appropriateness of empirical treatment recommendations: an international point-prevalence study

An accurate knowledge of the epidemiology of community-acquired pneumonia (CAP) is key for selecting appropriate antimicrobial treatments. Very few etiological studies assessed the appropriateness of empiric guideline recommendations at a multinational level. This study aims at the following: (i) describing the bacterial etiologic distribution of CAP and (ii) assessing the appropriateness of the empirical treatment recommendations by clinical practice guidelines (CPGs) for CAP in light of the bacterial pathogens diagnosed as causative agents of CAP. Secondary analysis of the GLIMP, a point-prevalence international study which enrolled adults hospitalized with CAP in 2015. The analysis was limited to immunocompetent patients tested for bacterial CAP agents within 24 h of admission. The CAP CPGs evaluated included the following: the 2007 and 2019 American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA), the European Respiratory Society (ERS), and selected country-specific CPGs. Among 2564 patients enrolled, 35.3% had an identifiable pathogen. Streptococcus pneumoniae (8.2%) was the most frequently identified pathogen, followed by Pseudomonas aeruginosa (4.1%) and Klebsiella pneumoniae (3.4%). CPGs appropriately recommend covering more than 90% of all the potential pathogens causing CAP, with the exception of patients enrolled from Germany, Pakistan, and Croatia. The 2019 ATS/IDSA CPGs appropriately recommend covering 93.6% of the cases compared with 90.3% of the ERS CPGs (p < 0.01). S. pneumoniae remains the most common pathogen in patients hospitalized with CAP. Multinational CPG recommendations for patients with CAP seem to appropriately cover the most common pathogens and should be strongly encouraged for the management of CAP patients.info:eu-repo/semantics/publishedVersio

Prevalence and Etiology of Community-acquired Pneumonia in Immunocompromised Patients

BACKGROUND: The correct management of immunocompromised patients with pneumonia is debated. We evaluated the prevalence, risk factors, and characteristics of immunocompromised patients coming from the community with pneumonia. METHODS: We conducted a secondary analysis of an international, multicenter study enrolling adult patients coming from the community with pneumonia and hospitalized in 222 hospitals in 54 countries worldwide. Risk factors for immunocompromise included AIDS, aplastic anemia, asplenia, hematological cancer, chemotherapy, neutropenia, biological drug use, lung transplantation, chronic steroid use, and solid tumor. RESULTS: At least 1 risk factor for immunocompromise was recorded in 18% of the 3702 patients enrolled. The prevalences of risk factors significantly differed across continents and countries, with chronic steroid use (45%), hematological cancer (25%), and chemotherapy (22%) the most common. Among immunocompromised patients, community-acquired pneumonia (CAP) pathogens were the most frequently identified, and prevalences did not differ from those in immunocompetent patients. Risk factors for immunocompromise were independently associated with neither Pseudomonas aeruginosa nor non-community-acquired bacteria. Specific risk factors were independently associated with fungal infections (odds ratio for AIDS and hematological cancer, 15.10 and 4.65, respectively; both P = .001), mycobacterial infections (AIDS; P = .006), and viral infections other than influenza (hematological cancer, 5.49; P &lt; .001). CONCLUSIONS: Our findings could be considered by clinicians in prescribing empiric antibiotic therapy for CAP in immunocompromised patients. Patients with AIDS and hematological cancer admitted with CAP may have higher prevalences of fungi, mycobacteria, and noninfluenza viruses

International prevalence and risk factors evaluation for drug-resistant Streptococcus pneumoniae pneumonia

Objective: Streptococcus pneumoniae is the most frequent bacterial pathogen isolated in subjects with Community-acquired pneumonia (CAP) worldwide. Limited data are available regarding the current global burden and risk factors associated with drug-resistant Streptococcus pneumoniae (DRSP) in CAP subjects. We assessed the multinational prevalence and risk factors for DRSP-CAP in a multinational point-prevalence study. Design: The prevalence of DRSP-CAP was assessed by identification of DRSP in blood or respiratory samples among adults hospitalized with CAP in 54 countries. Prevalence and risk factors were compared among subjects that had microbiological testing and antibiotic susceptibility data. Multivariate logistic regressions were used to identify risk factors independently associated with DRSP-CAP. Results: 3,193 subjects were included in the study. The global prevalence of DRSP-CAP was 1.3% and continental prevalence rates were 7.0% in Africa, 1.2% in Asia, and 1.0% in South America, Europe, and North America, respectively. Macrolide resistance was most frequently identified in subjects with DRSP-CAP (0.6%) followed by penicillin resistance (0.5%). Subjects in Africa were more likely to have DRSP-CAP (OR: 7.6; 95% CI: 3.34-15.35, p < 0.001) when compared to centres representing other continents. Conclusions: This multinational point-prevalence study found a low global prevalence of DRSP-CAP that may impact guideline development and antimicrobial policies. Published by Elsevier Ltd on behalf of The British Infection Association

Prevalence and risk factors for Enterobacteriaceae in patients hospitalized with community-acquired pneumonia

Background and objective Enterobacteriaceae (EB) spp. family is known to include potentially multidrug-resistant (MDR) microorganisms, and remains as an important cause of community-acquired pneumonia (CAP) associated with high mortality. The aim of this study was to determine the prevalence and specific risk factors associated with EB and MDR-EB in a cohort of hospitalized adults with CAP. Methods We performed a multinational, point-prevalence study of adult patients hospitalized with CAP. MDR-EB was defined when >= 3 antimicrobial classes were identified as non-susceptible. Risk factors assessment was also performed for patients with EB and MDR-EB infection. Results Of the 3193 patients enrolled with CAP, 197 (6%) had a positive culture with EB. Fifty-one percent (n = 100) of EB were resistant to at least one antibiotic and 19% (n = 38) had MDR-EB. The most commonly EB identified were Klebsiella pneumoniae (n = 111, 56%) and Escherichia coli (n = 56, 28%). The risk factors that were independently associated with EB CAP were male gender, severe CAP, underweight (body mass index (BMI) < 18.5) and prior extended-spectrum beta-lactamase (ESBL) infection. Additionally, prior ESBL infection, being underweight, cardiovascular diseases and hospitalization in the last 12 months were independently associated with MDR-EB CAP. Conclusion This study of adults hospitalized with CAP found a prevalence of EB of 6% and MDR-EB of 1.2%, respectively. The presence of specific risk factors, such as prior ESBL infection and being underweight, should raise the clinical suspicion for EB and MDR-EB in patients hospitalized with CAP